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COMMUNITY AND ECOSYSTEM ECOLOGY

No Detection of Cry1Ac Protein in Soil After Multiple Years ofTransgenic Bt Cotton (Bollgard) Use

GRAHAM HEAD, JAMES B. SURBER, JON A. WATSON, JOHN W. MARTIN, AND JIAN J. DUAN1

Monsanto Company, Ecological Technology Center, 800 North Lindbergh, St. Louis, MO 63141

Environ. Entomol. 31(1): 3036 (2002)

ABSTRACT Soil samples were collected from within and outside six elds where insect-resistanttransgenic cotton (Bollgard) encoding the Bacillus thuringiensis Berliner (Bt) subsp. kurstaki cry1Acgene had been grown and subsequently incorporated into soil by postharvest tillage for 36 consec-utiveyears. The level of Cry1Ac protein in these samples (collected 3 mo afterthe last seasons tillage)was evaluated using both enzyme-linked immunosorbent assays (ELISA) and bioassays with a

susceptible insect species, Heliothis virescens (F.), the tobacco budworm. Both methods revealed thatno detectable Cry1Ac protein was present in any of the soil samples collected from within or outsidethe Bollgard elds. Based on the results from reference standards, the limit of detection for the ELISAwas 3.68 ng of extractable protein per gram of soil, and that of the bioassay (measured by EC50) was8 ng of biologically active protein per gram of soil. Together, these ndings demonstrate that theamount of Cry1Ac protein accumulated as a result of continuous use of transgenic Bt cotton, andsubsequent incorporation of plant residues into the soil by postharvest tillage, is extremely low anddoes not result in detectable biological activity.

KEYWORDS Bacillus thuringiensis, transgenic cotton, Cry1Ac protein, environmental risk, enzyme-linked immunosorbent assays, insect bioassay

ADVANCEMENTS IN GENETIC engineering technologyhave enabledthe introduction and expression of novelgenes in plants to produce agronomically useful traitssuch as insect and disease resistance. Several speciesof crop plants (including cotton, corn, and potato)have been genetically modied to express genes ofvarious subspecies of Bacillus thuringiensis Berliner(Bt) encoding insecticidal proteins (-endotoxins)(see Lewellyn et al. 1994,Persley 1996, Federici 1998).These insecticidal proteins confer protection to theplant from damage by insect herbivores. The im-proveddelivery system of Bt toxinsthrough transgenicplants has great potential to reduce the reliance ontraditional chemical insecticides in insect pest controlprograms (e.g., Hoffmann et al. 1992). In addition,because Bt proteins are highly specic in their effects,the transgenic crop plants producing these proteinshave advantages over broad-spectrum pesticides infacilitating integration of other environmentally be-nign pest control strategies such as biological controlinto integrated pest management (IPM) programs(e.g., Bolin et al. 1996, Mascarenhas and Luttrell 1997,Orr and Landis 1997, Schuler et al. 1999, Reed et al.

2001).However, concerns have been raised over the en-

vironmental risks associated with the large-scale com-

mercial release of transgenic crops (see review inWilliamson 1992). These environmental concerns in-clude (1) risk of enhanced selection pressure for Bt-resistance in target insect pests, and (2) possible im-pacts on nontarget organisms that are of ecologicaland/or economic interest. One important aspect toconsider in evaluating these concerns is the possibleaccumulation and persistence of the plant-producedBt proteins in soils where the crops are repeatedlygrownandresidues of thecrop plantsareincorporated

into the soil by tillage or as litter.Recent laboratory studies (Venkateswerlu andStotzky 1992, Tapp et al. 1994, Tapp and Stotzky 1995,Crecchio and Stotzky 1998) have shown that insecti-cidal Cry proteins from B. thuringiensis subsp. kurstakiand subsp. tenebrionis are readily adsorbed at equilib-rium and bound to clay minerals and humic acids, andthat their insecticidal activity is maintained or en-hanced in the soil-toxin complexes. Crecchio andStotzky (1998) also showedthat under laboratory con-ditions, insecticidal protein (Cry1Ac) from B. thurin-giensis subsp. kurstaki, which had bound to soil humic

acids degraded more slowly than free protein. Basedon those laboratory ndings, these researchers hy-pothesized that incorporation of Bt proteins into soilfrom repeated large-scale useof transgeniccrop plantscould exceed the rate of natural degradation and in-activation, thereby leading to an accumulation of the

1 To whom correspondence should be addressed: e-mail:[email protected].

0046-225X/02/00300036$02.00/0 2002 Entomological Society of America


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protein in the soil that could reach biologically activelevels.

To date, however, this hypothesis has not beenevaluated under eld conditions. No studies have ex-amined the levels of Bt protein in soils of agriculturalelds where transgenic Bt crops have been repeatedlyplanted and residues of the crops incorporated intothe soil. In the current study, we use both insectbioassays and enzyme-linked immunosorbent assays(ELISA) to evaluate levels of Cry1Ac protein in soilswhere transgenic Bt cotton producing Cry1Ac proteinhad been continuously grown, and subsequently in-corporated into soil by postharvest tillage, for 36 yr.

Materials and Methods

Soil Samples. Soil samples were collected from sixdifferent sites where Bollgard cottonhadbeen plantedin each year for the previous 36 consecutive years.The history of Bollgard cotton growing at each sam-pling site is summarized in Table 1. In each year, all theBollgard elds were cultivatedoneto twotimes duringthe growing season, and the plant stalks were rstshredded with motor-driven shredders and then tilledinto the soil using disk plows at the end of each seasonimmediately following harvest of the cotton lint andseeds. All soil samples were collected in February1999, 3 mo after the last seasons tillage. At the time ofsampling, a majority of plant residues incorporatedinto the soil inthe previous seasons withshredders anddisk plowings (7.6 cm deep) had mostly decayed;thus, all the soil core samples collected at this timecontainedfully decayed plant residues, as well as somepartially decayed plant residues.

At each of the sampling sites, three core samples,each 15.2 cm deep and 7.6 cm in diameter, were ran-domly taken from the Bollgard cotton eld using abulb setter. The distance between each core samplewithin the same Bollgard eld ranged from 30.5 to

45.7 m. One soil sample also was taken from a point6.1 m outside of the Bt cotton eld at each site andserved as a negative control. These negative controlsites were either noncultivated areas or cultivatedeld plots where no Bt crops had been planted. Allcore samples were placed into glass jars, which were

then placed on dry ice and immediately sent to Mon-santo (St. Louis, MO) for evaluation. Although the pH(negative logarithm of hydrogen iron concentration)of the soil samples was not measured, all of the sampleswere collected from cotton elds in the main cottonbelt of the United States of America and the pH ofthese samples should be within the normal range forcotton growing regions.

Upon receipt at Monsanto, all soil samples werestored at 80C before any analysis. Before analysis byELISA and insect bioassay, each soil core sample wasground using a mortar and pestle and then homoge-nized using an electric motor driven homogenizer (at10,000-40,000 revolutions per minute) for at least 15min. These procedures allowed all components (suchas soil particles and decayed plant residues) of eachcore sample to be fully mixed and homogenized forELISA and insect bioassay.

TestingProcedures. Twodifferent methods (ELISA

and bioassays with a susceptible insect species) wereused to determine the level of Cry1Ac protein in soilsamples. While ELISA allows the quantication ofboth functional and nonfunctional Cry1Ac protein insoil based on levels of protein binding to an antibody,bioassays allow assessment of the level of bio-activeprotein in the soil. In addition, the efciency of de-tection by ELISA depends on the amount of theCry1Ac protein that can be extracted from the soil,whereas the level of biological activity detected bybioassay is determined by the insects ability to extractCry1Acprotein from thesoil (Tapp andStotzky 1995).

ELISA. The ELISA used in this study involved twostages: protein extraction and quantication. To ex-tract Cry1Ac protein from the soil samples, the buffersolution described by Palm et al. (1994), consisting of50 milimolar sodium borate (pH 10.5), 0.75 molarpotassium chloride, 0.075% polyoxyethylenesorbitanmonolaurate (Tween 20), and 10 mM ascorbic acid,was used. The potassium chloride and Tween 20 wereused in the buffer solution to maximize extractionefciency by discouraging potential ionic and hydro-phobic interactions between soilparticles and Cry1Acprotein (Palm et al. 1994). Soil samples were mixedwith the buffer solution at a ratio of 1:2, and homog-enized for 15 s using a Brinkmann Polytron PT3000(Brinkmann Instruments, Westbury, NY). Immedi-ately after homogenization, the soil-buffer mixturewas centrifuged at 16,000 g for 10 min. After cen-trifugation, an aliquot was removed from the super-natant and analyzed using a double-antibody sand-wich ELISA as described by Fuchs et al. (1990) andPalm et al. (1994). The amount of soil extracted fromeach core sample for analysis ranged from 0.5052 to0.6112 g.

For quantication of Cry1Ac protein in test sam-ples, the wells of a microtiter plate (F-96 MaxiSorb,

Nunc, Roskilde, Denmark) were rst coated with aprimary monoclonal anti-Cry1Ac antibody (Mon-santo, lot number M19N4A6). A secondary polyclonalrabbit anti-Cry1Ac antibody (Monsanto, lot numberMR129) and the test sample (aliquot extract) thenwere added to the plate, and the plate was incubated

Table 1. History of Bt cotton planting at different sampling

sites

Sampling site Soil type

Years ofBt cotton

grown prior tosampling

Loxley Ag Center, BelleMina, AL

Sandy loam 19961998

Davis Ag Consulting,Montgomery, AL

Sandy loam 19961998

Lecroix Farms, Belle Mina,AL

Sandy loam 19951998

USDA-ARS, MississippiStation, MS

Silt loam 19941998

Hood-Farms, Gunnison, MS Silt loam 19941998Delta and Pine, Scott, MS Silt loam 19931998

February 2002 HEAD ET AL.: NO CRY1AC PROTEIN IN SOIL AFTER YEARS OF BT COTTON USE 31


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for 1 h at 37C. The reaction volume was 250 l foreach well. Extracts were diluted at least 10 times with0.5% (weight/volume) ovalbumin phosphate buffersolution to minimize potentialmatrix effects. After the1-h incubation, the plate was washed using the phos-phate buffer solution and then treated with a donkeyanti-rabbit alkaline phosphate conjugate antibody. Af-ter the treatment, the plate was incubated again for 1 hand subsequently washed with the phosphate buffersaline. The plate then was treated with p-nitrophenylphosphate, incubated at room temperature for about30 min, and then analyzed by spectrophotometry at405 nm, with a reference wavelength of 655 nm, in aBio-Rad model 3350 microtiter plate reader (Bio-Rad,Hercules, CA). For purposes of quantication, a sev-en-point standard curve comprising puried Cry1Acstandard protein was included in each microplate.Absorbance readings and Cry1Ac standard proteinconcentrations were logarithmically transformed and

tted with a quadratic regression model for extrapo-lation and estimation of Cry1Ac protein levels in soilstaken from within and outside of Bt cotton elds. Thelimit of detection was calculated as the mean back-ground Cry1Ac protein level in soils from non-Bt con-trol samples (two assays for each of the six soil sam-ples) plus three standard deviations.

The Cry1Ac protein standard (lot number: 11498BR 1098) employed in this study was produced atMonsanto using the Bacillus thuringiensis spp. kurstakiHD-73 strain according to procedures described inMacIntosh et al. (1990). Puried protein (containing

94% Cry1Ac) was trypsinized and solubilized in 100mM sodium carbonate buffer, pH 10.5, before use indifferent assays. To determine the efciency ofCry1Ac protein extraction from different soil samples,soil from non-Bt control samples was spiked with theCry 1Ac standard protein at a rate of 100 ng/g of soil(ppb), andthen analyzed using the ELISA proceduresdescribed above.

Bioassay. The larvae of tobacco budworm, Heliothisvirescens (F.), were used in the bioassay because oftheir highsusceptibility to Cry1Ac protein(Hofte andWhitely 1989; Sims and Holden 1996). Eggs of H.virescens were purchased from Ecogen (Langhorne,PA) and shipped to Monsanto by express mail in aStyrofoam cooler that contained cooling packs inside.Upon receipt of the shipment, eggs were immediatelyplaced in a ventilated plastic box, which was thenplaced in a growth chamber (Percival, Boone, IA)with controlled climatic conditions (27 1.5C, 50 10% RH) for incubation.

To assay for Cry1Ac protein, soil samples were in-corporated into the articial diet, and then presentedto rst-instar H. virescens. The bioassay procedure wassimilar to that previously described by Sims andHolden (1996). One gram of soil from each core sam-

ple was thoroughly slurried with 4 ml water in a 50-mlcentrifuge tube on a Vortex Mixer, and then mixedwith agar-based liquid diet to bring it up to a totalvolume of 20 ml. The diet used in the bioassay was astandard soybean-based multiple specieslepidopterandiet (King and Hartley 1992) and was purchased from

Southland Products (Lake Village, AR). The soil-dietmixture for each soil sample was added to 16 cells ofa bioassay tray (C-D International, Pitman, NJ) at arate of 1 ml per cell using a repeater pipettor. Eachbioassay tray contained a total of 128 cells so that eightdifferent test samples (each with 16 cells) were runsimultaneously on each tray. After the soil-diet mix-ture cooled and solidied, one rst-instar H. virescens(1224 h old) was introduced into each of the cells ofthe bioassay tray using a camels hair brush. The bio-assay tray was sealed with vented transparent acetatecovers, and placed in a growth chamber at 27C and3040% RH for 7 d. After the 7-d incubation, survivaland mass of H. virescens larvae for each test samplewere determined.

To serve as a reference standard, non-Bt soil sam-ples (from Loxley Agricultural Center, Loxley, AL)were spiked with a series of 10-fold dilutions of pureCry1Ac protein. The series of concentrations used as

a reference standard were prepared by thoroughlymixing 1 g of the non-Bt soil with 2 101, 2 102, 2103, 2 104, o r2 105 ng Cry1Ac in 4 ml water, whichthen wasmixed with agar-based liquid H. virescens dietto bring it up to a total volume of 20 ml. The nalconcentrations of Cry 1Ac in the soil-diet mixturewere 1 100, 1 101, 1 102, 1 103, and 1 104

ng per ml of the mixture. The soil-diet mixtures for thereference standard were assayed with H. virescensusing the procedures described previously for test soilsamples. Non-Bt soil collected from the same site andnot spiked with Cry1Ac protein was used as a negative

control.Analysis of variance (ANOVA) was used to analyzefor differences in survival and mass of H. virescensexposed to soil samples collected within and outsidetheBt cottonelds at different locations. Because onlyone soil sample was collected from outside the Bt eldat each of the sites, the interaction between samplesite and Bt treatment was not examined. All statisticalanalyses were performed with JMP Statistical Discov-ery Software (SAS Institute 1995). Probit analysis wasused to analyze the doseresponse relationships be-tween the standard Cry1Ac concentrations spiked insoil and the mortality of the test H. virescens larvae(SAS Institute 2000), while a three-parameter logisticregression model (Sims and Holden 1996) was used toanalyze the doseresponse relationships between thestandard Cry1Ac concentrations spiked in the soil andthe mass of surviving H. virescens (SAS Institute 1995).

Results

Quantification by ELISA. No Cry1Ac protein wasdetected by ELISA in any of thesoil samples collectedeither within or outside the Bollgard elds at the sixsites. When soil from outside the Bt eld (i.e., non-Bt

treatment) at each site was spiked with a knownamount (100 ng) of pure Cry1Ac protein, a mean(SD) of 31.76% (6.72) of the originally spikedCry1Ac protein was detected by ELISA (Table 2).Additional tests with the negative control samplesfurther established that the mean (SD) background

32 ENVIRONMENTAL ENTOMOLOGY Vol. 31, no. 1


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level of Cry1Ac protein in the six non-Bt soil sampleswas 2.36 (0.44) ng of extractable protein per gramsoil. The limit of detection for Cry1Ac protein in soilbased on three standard deviations was 3.68 ng ofextractable protein per gram of soil. Based on theaverage extraction(orrecovery) rate of 31.76%(Table2), the level of Cry 1Ac protein that could be detectedby ELISA would be 11.57 ng/g of soil.

Biological Activity Determined by Bioassay. Thedoseresponse relationships between the standardconcentration of Cry1Ac protein spiked in soil andmortality and growth of H. virescens larvae are pre-sented in Fig. 1 (A and B). Probit analysis of dose-mortality relationship indicates that the concentrationof Cry1Ac protein spiked into soil required to kill 50%ofH. virescens larvae (LC50) was 755 ng/g of soil (withthe 95% condence interval being from 312 to 1,863ng/g). Based on the logistic regression model, theconcentration required to reduce larval growth by50% (EC50) (relative to the negative control) wasestimated to be 8 ng/g of the soil (with the 95%condence interval being from 7 to 9 ng/g). Thus, anyconcentrations of Cry1Ac protein in soil at or above8 ng/g of soil (corresponding to 0.4 ng/ml of diet ofsoil mixture) should be effectively detected by theH. virescens bioassay.

For test samples, the mean survival (SE) (acrossall sample sites) was 97.7% (0.20) for soil samplescollected from Bt cotton elds, and 98.0% (0.30) forsamples from outside the Bt cotton elds (Fig. 2A).

The mean mass (SE) of surviving H. virescens larvaewas 107.8 mg (2.23) and 99.8 mg (4.23) for soilsamples collected from within and outside Bt cottonelds, respectively (Fig. 2B). ANOVA detected nosignicant differences in either survival or mass ofH. virescens larvae exposed to soil samples from within

Fig. 1. Reference standard doseresponse ofH. virescenslarvae feeding on diet mixed with soil samples spiked withknown doses (2 101, 2 102, 2 103, 2 104, and 2 105

ng)of pureCry1Acproteinper gramsoil. (A)Doseresponsefor larval survival, and (B) doseresponse for larval mass(mg).

Fig. 2. Response ofH. virescens larvae to diet mixed withsoil samples collected from within and outside transgenic Btcotton eldsat six different sites. (A) Larval survival,and(B)

average mass (mg) of surviving larvae.

Table 2. Efficiency of ELISA in detection of Cry1Ac protein

spiked in soils and background level of Cry1Ac protein in non-Bt

soil from different sampling sites

Sampling site

Levels of Cry1Acprotein in soil

samples spiked ata rate of

100 ng/g soil

Background level ofCry1Ac protein

(ng/g soil) in non-Bt soil samples

Loxley Ag Center, BelleMina, AL

38.89 2.61

Davis Ag Consulting,Montgomery, AL

35.63 2.81

Lecroix Farms, Belle Mina,AL

29.70 2.62

USDA-ARS, MississippiStation, MS

38.07 1.59

Hood Farms, Gunnison, MS 23.94 2.16Delta and Pine, Scott, MS 24.34 2.34Mean SD 31.76 6.72 2.36 0.44



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and outside Bt cotton elds, nor any signicant dif-ferences among different sampling sites (Tables 3 and4). If anything, there was a nonsignicant trend to-ward larger masses for larvae exposed to the soil sam-ples from within the Bt cotton elds. When the doseresponse relationships between the standard Cry1Acprotein concentrations spiked in soil and mortality orgrowth (mass) of H. virescens larvae are considered(Fig. 1 A and B), these results indicate that levels of

Cry1Ac protein in the soil from Bt cotton elds are nothigh enough to be biologically active against even ahighly susceptible insect, H. virescens.

Because the non-Bt soil samples assayed in thisstudywerecollected from near(6.1 m away) Bt cottonelds, one might argue that the non-Bt soil samplescould be contaminated with Cry1Ac proteins from theBt cotton elds. However, mean larval survival (97.798.0%) and mass (98.8107.8 mg) ofH. virescens lar-vae assayed for both non-Bt and Bt soil treatments inthis study were within the normal ranges for the sur-vival (95100%) and growth (94157 mg) of 7-d old H.

virescens larvae established previously using the samearticial diets with no soil or Bt protein present (J.J.D.and J.W.M., Monsanto, unpublished data).

Discussion

Results from both ELISA and insect bioassay indi-cated that repeated agricultural use of transgenic Btcotton (Bollgard) expressing Cry1Ac protein did notresult in detectable levels of Cry1Ac protein in soil.The amount of Cry1Ac protein, if any, accumulatedfrom 3 to 6 yr of repeated use of the transgenic cottonand subsequent incorporation of the plant residuesinto the soil by tillage was below the limit of detectionfor both ELISA and bioassay. Thus, these ndings donot support the assertions that repeated and large-scale use of transgenic crop plants could lead to ac-cumulation and/or persistence of Bt proteins in soil atlevels that would result in biological impacts (Tappand Stotzky 1995, Crecchio and Stotzky 1998).

The limits of Cry1Ac detection by both ELISA andbioassay used in this study were determined with soil

samples spiked with pure Cry1Ac protein, and werenot validated with transgenic Bt cotton plant residues.However, previous studies by Palm et al. (1994, 1996)demonstrated that the protein extraction procedureand ELISA (comparable to that used here) workedeffectively to detect Cry1Ac either spiked in soil or asa component of transgenic Bt plant tissues incorpo-rated into soil. The efciency of Cry1Ac extractionreported by Palm et al. (1994) ranged from 27 to 60.2%for proteinspiked in different types of soil and 1770%for protein as a component of different cultivars oftransgenic Bt cotton leaf tissues. In the study reportedhere, the rate of pure Cry1Ac protein recovered fromspiked soil samples was 24.3438.89% with a mean of31.76% (Table 2). Based on this range of extractionefciencies and the detection limit of 3.68 ng extract-able protein per gram of soil, the level of Cry1Acprotein that could have beendetected by ELISA insoilsamples collected from the six different eld sites

ranged from 9.46 to 15.14 ng/g of soil (with a mean of11.57 ng/g).

A previous study (MacIntosh et al. 1990) showedthat H. virescens larvae were extremely susceptible topure Cry1Ac incorporated in the larval diet, and theEC50 against H. virescens larvae was 1.3 ng of Cry1Acprotein per ml of insect diet. The bioassay in the studyreported here showed that the EC50 for pure Cry1Acspiked in soil was 8 ng/g of soil (corresponding to 0.4ng of Cry1Ac protein per milliliter of diet-soil mix-ture). These ndings indicate that the H. virescensbioassay is highly sensitive in detecting the biological

activity of Cry1Acproteinincorporated with their dietat the EC50 level. Although the efciency of Cry1Acextraction by H. virescens larvae from various dietmixtures has never been quantied partly because ofthe technical difculty, there have been no publishedstudies suggesting that H. virescens larvae would dif-ferentially extract Cry1Ac protein from spiked soilversus transgenic Bt plant materials when those ma-terials are incorporated into the larval diet. In fact,previous studies strongly suggested that lepidopteranlarvae (including H. virescens) show comparable re-sponses to pure Bt proteins spiked in soil (MacIntoshet al. 1990, Sims and Holden 1996, Crecchio andStotzky 1998) and Bt protein as a component of trans-genic Bt plant tissues (Ream et al. 1994).

Based on an average expression rate of 20 g ofCry1Ac protein per gram of dry Bollgard cotton planttissue (Kollwyck and Hamilton 1999), a conservativeestimate of the level of Cry1Ac protein added to thetop three inches of soil in Bollgard elds (with 60,000plants per acre, each plant250 g dry weight) for eachgrowing season would be 650 ng/g of dry soil. If allof the Bt protein accumulated in the soil without anydegradation and/or inactivation, the level of Cry1Acprotein after 6 yr (seasons) of continuously planting

Bollgard cotton would be 252 and 195 times higherthan the limits of detection by ELISA and bioassayused in this study, respectively. Furthermore, this es-timate does not include the amount of the Cry1Acprotein (if any) that may be released into soil bygrowing plants through root exudates. Recently, Sax-

Table 3. ANOVA table for H. virescens larval survival

Variable df Sum ofsquares

F ratio P F

Sample site 5 0.1941 1.1352 0.3937Bt treatment 1 0.0015 0.0438 0.8378Sample site Bt treatment 5 0.1245 0.7286 0.6153Error 12 0.4103

Square root transformation was applied to data prior to analysis.

Table 4. ANOVA table for H. virescens larval mass

Variable df Sum ofsquares

F Ratio PF

Sample site 5 695.0460 1.7772 0.1921Bt treatment 1 285.8789 3.6549 0.0801Sample site Bt treatment 5 398.7850 1.0197 0.4483Error 12 938.6227



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ena et al. (1999) reported that transgenic corn plantsexpressing the cry1Ab gene release Cry1Ab proteininto soil through root exudation. No matter what thesource of Cry1Ac, because the soil samples in thestudy reported here were taken 3 mo after the lastgrowing season, the absence of detectable Cry1Acprotein in all of the soil samples suggest both little orno accumulationover years andrapid breakdownaftereach season (i.e., reduced to undetectable levels in 3mo). Future studies should examine Cry1Ac proteindissipation in Bt crop elds at different times in thegrowing season, as well as immediately after incorpo-ration of plant residues into the soil after harvest. Suchstudies will assist us in assessing the potential impactof Bt plant residues on soil dwelling nontarget organ-isms.

The persistence of Bt proteins in the environmentis a function primarily of (1) the concentration added,and (2) the rate of inactivation and degradation by

both biotic and abiotic factors; when the rate of ad-dition is faster than the rate of inactivation and/ordegradation, the toxin accumulates (Venkateswerluand Stotzky 1992, Tapp and Stotzky 1995, Crecchioand Stotzky 1998). Palm et al. (1996) showed that,when incubated in soil under laboratory conditions,Cry1Ac protein contained in transgenic Bt cottonplant residues degraded in soil at a rate similar to orgreater than pure Cry1Ac proteins. Using insect bio-assays, Ream et al. (1994) estimated that, at roomtemperature (24C), the half-life of Cry1Ac proteinwas 9.320.2 d when spiked into silt loam soil, and 41 d

when added to soil as transgenic Bt cotton plant tis-sues. Studies of the environmental fate of other Btproteins, including those used in transgenic Bt potatoand Bt corn (Cry3Aa and Cry 1Ab), have found thatthe half-lives of these proteins in soil are generally lessthan 20 d (Ream et al. 1994, Sims and Holden 1996,Palm et al. 1996). Although degradation of Bt Cryprotein in soil does not follow a rst order process andthe rate of degradation could become slower as theamount of the Cry protein becomes less in soil (e.g.,Palm et al. 1996), results from the current study dem-onstrate that the amount of Cry1Ac protein accumu-lated as a result of the continuous use of transgenic Btcotton and subsequent incorporation of the plant res-idues into the soil by postharvest tillage in multipleseasons, does not result in detectable immunologicaland biological activity. The apparently rapid break-down of other Bt proteins in soil, at rates comparableto that measured for Cry1Ac (Ream et al. 1994, Palmet al. 1996, Sims and Holden 1996), suggests that theseproteins also will not be accumulating at biologicallysignicant levels.

Acknowledgments

We thank Gregg Dixon, Zachary Shappley, and Alan Weirfor assistance in providing us soil samples. Tom Nickson andMike Mckee (Ecological Technology Center, Monsanto)provided helpful comments on an earlier draft of the manu-script. Changjian Jiang (of the Environmental Science Cen-ter, Monsanto) provided assistance in statistical analysis.

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Received for publication 5 October 2000; accepted 12 Oc-tober 2001.


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